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The naturally occurring nucleotide 2-deoxy-adenosine 5'-triphosphate (dATP) can be used by cardiac muscle as an alternative energy substrate for myosin chemomechanical activity. We and others have previously shown that dATP increases contractile force in normal hearts and models of depressed systolic function, but the structural basis of these effects has remained unresolved. In this work, we combine multiple techniques to provide structural and functional information at the angstrom-nanometer and millisecond time scales, demonstrating the ability to make both structural measurements and quantitative kinetic estimates of weak actin-myosin interactions that underpin sarcomere dynamics. Exploiting dATP as a molecular probe, we assess how small changes in myosin structure translate to electrostatic-based changes in sarcomere function to augment contractility in cardiac muscle. Through Brownian dynamics simulation and computational structural analysis, we found that deoxy-hydrolysis products [2-deoxy-adenosine 5'-diphosphate (dADP) and inorganic phosphate (Pi)] bound to prepowerstroke myosin induce an allosteric restructuring of the actin-binding surface on myosin to increase the rate of cross-bridge formation. We then show experimentally that this predicted effect translates into increased electrostatic interactions between actin and cardiac myosin in vitro. Finally, using small-angle X-ray diffraction analysis of sarcomere structure, we demonstrate that the proposed increased electrostatic affinity of myosin for actin causes a disruption of the resting conformation of myosin motors, resulting in their repositioning toward the thin filament before activation. The dATP-mediated structural alterations in myosin reported here may provide insight into an improved criterion for the design or selection of small molecules to be developed as therapeutic agents to treat systolic dysfunction.

The formation of toxic Amyloid β-peptide (Aβ) oligomers is one of the earliest events in the molecular pathology of Alzheimer's Disease (AD). These oligomers lead to a variety of downstream effects, including impaired neuronal signaling, neuroinflammation, tau phosphorylation, and neurodegeneration, and it is estimated that these events begin 10 to 20 y before the presentation of symptoms. Toxic Aβ oligomers contain a nonstandard protein structure, termed α-sheet, and designed α-sheet peptides target this main-chain structure in toxic oligomers independent of sequence. Here we show that a designed α-sheet peptide inhibits the deleterious effects on neuronal signaling and also serves as a capture agent in our soluble oligomer binding assay (SOBA). Pre-incubated synthetic α-sheet-containing Aβ oligomers produce strong SOBA signals, while monomeric and β-sheet protofibrillar Aβ do not. α-sheet containing oligomers were also present in cerebrospinal fluid (CSF) from an AD patient versus a noncognitively impaired control. For the detection of toxic oligomers in plasma, we developed a plate coating to increase the density of the capture peptide. The proof of concept was achieved by testing 379 banked human plasma samples. SOBA detected Aβ oligomers in patients on the AD continuum, including controls who later progressed to mild cognitive impairment. In addition, SOBA discriminated AD from other forms of dementia, yielding sensitivity and specificity of 99% relative to clinical and neuropathological diagnoses. To explore the broader potential of SOBA, we adapted the assay for α-synuclein oligomers and confirmed their presence in CSF from patients with Parkinson's disease and Lewy body dementia. [ABSTRACT FROM AUTHOR]

Transthyretin, β2-microglobulin, lysozyme, and the prion protein are four of the best-characterized proteins implicated in amyloid disease. Upon partial acid denaturation, these proteins undergo conformational change into an amyloidogenic intermediate that can self-assemble into amyloid fibrils. Many experiments have shown that pH-mediated changes in structure are required for the formation of the amyloidogeneic intermediate, but it has proved impossible to characterize these conformational changes at high resolution using experimental means. To probe these conformational changes at atomic resolution, we have performed molecular dynamics simulations of these proteins at neutral and low pH. In low-pH simulations of all four proteins, we observe the formation of a-pleated sheet secondary structure, which was first proposed by 1. Pauling and R. B. Corey [(1951) Proc Natl. Acad. Sci. USA 37, 251-256]. In all β-sheet proteins, transthyretin and p2-microglobulin, a-pleated sheet structure formed over the strands that are highly protected in hydrogen-exchange experiments probing amyloidogenic conditions. In lysozyme and the prion protein, a-sheets formed in the specific regions of the protein implicated in the amyloidogenic conversion. We propose that the formation of α-pleated sheet structure may be a common conformational transition in amyloidosis. [ABSTRACT FROM AUTHOR]

Here, we describe the folding/unfolding kinetics of α[SUB3]D, a small designed three-helix bundle. Both IR temperature jump and ultra-fast fluorescence mixing methods reveal a single-exponential process consistent with a minimal folding time of 3.2 ± 1.2 μs (at ≈ 50°C), indicating that a protein can fold on the 1- to 5-μs time scale. Furthermore, the single-exponential nature of the relaxation indicates that the prefactor for transition state (TS)-folding models is probably ≥ 1 (μs)1 for a protein of this size and topology. Molecular dynamics simulations and IR spectroscopy provide a molecular rationale for the rapid, single-exponential folding of this protein. α[SUB3]D shows a significant bias toward local helical structure in the thermally denatured state. The molecular dynamics-simulated TS ensemble is highly heterogeneous and dynamic, allowing access to the TS via multiple pathways. [ABSTRACT FROM AUTHOR]

Examines on the flexible thioether linker as a loop mimetic in the human yes kinase-associated protein (YAP 65) WW domain. Comparison between amino acid and glycine; Analysis of the influence of loops on folding kinetics and stability; Effect of thioether linker on global fold of the domain.

Explores mapping of the early steps in the pH-induced conformational conversion of the prion protein (PrP). Factors that trigger conversion of the PrP; Effect of acidic pH on PrP conformation; Characterization of change at the atomic level.

Focuses on a study that presented multiple native and denaturation simulations of the B and E domains of the three-helix bundle staphylococcal protein A from Staphylococcus aureus. Methodology of the study; Results and discussion; Conclusion.

Here, we describe the folding/unfolding kinetics of alpha3D, a small designed three-helix bundle. Both IR temperature jump and ultrafast fluorescence mixing methods reveal a single-exponential process consistent with a minimal folding time of 3.2 +/- 1.2 micros (at approximately 50 degrees C), indicating that a protein can fold on the 1- to 5-micros time scale. Furthermore, the single-exponential nature of the relaxation indicates that the prefactor for transition state (TS)-folding models is probably >or=1 (micros)-1 for a protein of this size and topology. Molecular dynamics simulations and IR spectroscopy provide a molecular rationale for the rapid, single-exponential folding of this protein. alpha3D shows a significant bias toward local helical structure in the thermally denatured state. The molecular dynamics-simulated TS ensemble is highly heterogeneous and dynamic, allowing access to the TS via multiple pathways.